The interaction between PII and NAGK regulates arginine biosynthesis in the green microalga Haematococcus pluvialis

PII protein is widely acknowledged to regulate intracellular nitrogen and carbon metabolism by interacting with several crucial proteins. N-acetyl-L-glutamate kinase (NAGK), a rate-limiting enzyme for arginine biosynthesis, is regarded as a potential target of PII protein. Nevertheless, the regulatory function remains ambiguous in green algae and has not been investigated in Haematococcus pluvialis. In this study, the NAGK enzyme and PII protein of H. pluvialis (designated as HpNAGK and HpPII, respectively) and their interaction relationships were characterized. The results indicated that HpNAGK showed high similarity with the same enzyme in the green algae. A subcellular localization assay indicated that both HpPII and HpNAGK were located in the chloroplasts. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays distinctly verified the interaction between HpPII and HpNAGK, which occurs in the chloroplasts. The structure of the HpPII-HpNAGK complex was predicted through docking analysis. Moreover, the HpNAGK activity was significantly enhanced by HpPII in the presence of glutamine in vitro. Under nitrogen starvation, HpNAGK activity declined in vivo, concomitant with a reduction in arginine accumulation. The regulatory function of HpPII on HpNAGK activity aligned with that in Chlamydomonas reinhardtii but differed from that in Dunaliella salina, suggesting species specificity among green algae. These findings provide insights into the regulatory function of PII protein in green algae and help to unveil the response mechanisms of H. pluvialis to different nitrogen statuses.